Isolation of a Lectin from Apple Pollen - Dissertation Example

? Isolation of a Lectin from apple pollen Introduction: Lectins are glycoprotein present in the plants and animals. These Lectins are found in seeds,barks, fungi, plant roots, sea weeds and sponges, mollusks, fish eggs and body fluids of invertebrates. Lectin is involved in the processes such as transport of carbohydrates, cell wall elongation, cell-cell interaction, membrane receptor recognizers, storage proteins, growth regulation, self incompatibility and also functions as enzymes. The plant Lectin is used in many areas of biotechnology. The therapeutic use of the lectin is increasing day by day. Lectin offers a great opportunity to explore the carbohydrate- protein interactions occurring naturally in the system.(Van Damme, 1998). (Brooks et al., 1997). So a study on biochemical, physiological and molecular levels is required to explore the importance of Lectin in the plant cell. Lectin is present in many forms in the plant cells. The pollen grains of the apple are found to have some group of Lectin. These Lectin are heterodimers and have a great affinity for the glycopeptides with a molecular weight of 30,000 Daltons and have two subunits with N-acetyl glucosamine residues. Arabinose is found linked to the polypeptide chain as ?-L-arabinofuranosides through the hydroxylproline residues. (Sharon and Lis, 2007). The glycopeptides are found to contain lesser amount of serine, S-carboxyl methyl cysteine and other amino acids such as glycine as major amino acids. Cysteine (16%), Hydroxyproline (11%) and Glycine (12%) is the major amino acid composition of lectin. Lectin agglutinates the cells and precipitates the complex carbohydrates. The lectin has Arabinose monosachharide (93%) and Galactose (7.5%) of the total 37% carbohydrate content of lectin. (Sharon and Lis, 2007). These Lectins have greater usage in the medicine and medical field, as a biochemical tool, as a biological warfare, for studying carbohydrate recognition in the proteins etc., The Concavallin A is one of the most important lectin that is used in the affinity chromatography columns. Purification of Lectin from the pollen: Lectin is a protein having carbohydrate moiety. The first thing that we must look upon is whether it is an intracellular product or extra cellular product. Second thing to look upon is whether it is bound to any organelle or free protein. The Ammonium sulfate precipitation can be carried out after cell lysis. The cell disruption can be performed in many ways. Using mortar and pestle, sonication are some of the simple steps that can be easily practiced in lab. (Scopes, 1994).The cells are crushed using the saline buffer in order to maintain the pH. The protein extraction procedures are carried out in a cold room usually at 4 °C, in order to preserve the active part of the protein. The Ammonium sulfate precipitation is then carried out. Ammonium sulfate precipitation is a salting out process where the proteins are precipitated by adding ammonium sulfate at saturation constant. The saturation level varies for every protein and hence this phenomenon is widely used for the separation of the protein from the cell debris. The percentage of purification achieved at this stage is around 30 – 40%. Ammonium sulfate precipitation method is repeated to increase the concentration of the protein in the sample. After ammonium sulfate precipitation, the ammonium sulfate salt is removed from the protein by using the neutralizing buffer. The dialysis is the next downstream processing step. Dialysis is done using a semi-permeable membrane bag that has a very small pore size of 20 µm. Dialysis is done overnight in the saline to concentrate the protein. For further purification of the protein, chromatographic techniques are used. Affinity chromatography, anion exchange chromatography, gel permeation chromatography are some of the most commonly used chromatographic techniques for this protein. Same type of lectin is present in seeds and pollen grains hence the same procedure for the seed lectin extraction can be followed for the pollen lectin extraction. The pollen was grinded with phosphate buffer saline at 4 °C and kept overnight. The solution was centrifuged at 17000 rpm for 30 min and the supernatant was collected in a separate tube. (Joubert et al., 1986).The procedure was repeated again and the supernatant was collected. Now the supernatants of the two extractions were mixed together and used for further analysis. The glycoprotein was precipitated using the ammonium sulphate precipitation method for 65% saturation at 4 °C. (Joubert et al., 1986). After saturation, the solution was left overnight and the pellet was collected by centrifugation at 9000 rpm for 20 min. The pellet was suspended in the phosphate buffer saline and further purification was carried out. The dialysis bag was activated with the same buffer and dialysis was performed using the buffer first followed by water. The suspension obtained after dialysis was centrifuged at 9000 rpm for 20 min and the pellet was freeze dried for further analysis. The column chromatography and gel filtration chromatography was carried out for further purification of the sample. (Joubert et al., 1986). Affinity chromatography separates the proteins based on the reversible interaction between the protein and the specific ligand that is attached to the chromatographic matrix. (Baues and Gray, 1976). Affinity chromatography provides high selectivity, high resolution and high capacity for the proteins of interest. Here the target proteins bind very specifically and reversibly to the ligand. The unbounded materials are washed away from the column. The bounded protein s then desorbed from the column by using a competitive ligand. Desorption can also be performed by changing the polarity, pH and the ionic strength of the solution. Some of the disadvantages of affinity chromatography is that they have low binding affinity for the carbohydrate or polysaccharides and the difficulties in the synthesizing of the carbohydrate haptens. (Bonner, 2007). So because of these difficulties, the polyacrylamide gels were used for the purification. Because of the difficulties faced in this method some alterations were done for coupling the reduced disaccharides. The cyanoborohydride anion is used along with the Schiff base to reduce the protein. (Baues and Gray, 1976). Gel filtration chromatography is also used for the purification of the protein. Gel filtration chromatography separates the proteins based on their molecular size. This technique is more suited for the polishing of the proteins. Here the samples are eluted isocratically (i.e., with a single buffer with no gradient in pH). (Bonner, 2007). The most important thing to be noted in gel filtration chromatography is the column packing. Good column packing is required for the resolution of the protein. The length of the column should be as high as possible to ensure complete purification. The buffer used should be very compatible for the protein stability and activity. Anion exchange chromatography can be carried out next to the affinity chromatography. DEAE column can be used along with TBS-Ca equilibration. The column is washed with TBS-Ca and eluted with the step wise gradient of NaCl. The protein concentration of each fraction was found by using the agglutination assay. (Birck et al., 2004). The lectin activity assay was performed using trypsin -treated human erythrocytes of the O blood group. These erythrocyte solutions were diluted with TBS-Ca and are mixed with or without the inhibitors. Diluted MCL was also added to the solutions. The reaction mixture was shaked well for 10 minutes and the haemagglutination was observed for the samples. To know the factors that will affect the lectin, the pH was varied and its effect was evaluated at room temperature. The metal ion dependency was also estimated for Calcium, magnesium and Zinc ions. The molecular weight of the protein was estimated using the SDS –PAGE. The polyacrylamide gel electrophoresis is the most widely used technique for the determination of the molecular weight of the protein. SDS Page method is the universally used method for analyzing the mixture of proteins according to their respective size. (Desai et al.,1981). Separation of the proteins does not occur with similar charge. Therefore the proteins are treated with ionic detergent called SDS before the start and during the course of the electrophoresis. On the basis of the mass but not the charge the molecules are separated.(Scopes, 1994).The gel acts as a molecular sieve and hence separates the proteins on the basis of their size. The gel also suppresses the conventional current produced by the small temperature gradient which improves the resolution. The proteins are denatured and have a negative charge with a uniform charge to mass ratio when treated with SDS. Proteins migrate towards the anode at alkaline pH provided by the phosphate buffer through PAGE gel. The smaller peptides move faster followed by the larger peptides. Molecular weight of the separated protein can be analyzed by comparing the molecular weight of the standard protein and its mobility. (Dubey, 2006).Native PAGE is used to determine whether there are any isomers or not. Sometimes many isomers may be found together. This can be easily identified using native PAGE. The standard markers are used to determine the molecular weight. The ladder starting from 12 kb to 10 kb and even more molecular weight is also used. (Bulgakov, 2003). The subunits of the protein can also be found using the SDS-PAGE method. The lectin was denatured and the 10% poly acryl amide gel electrophoresis was performed. Both native PAGE and SDS-PAGE were performed for the given sample. The molecular weight of the given sample was identified using the molecular markers of high and low molecular weight. (Bulgakov, 2003) The Sepharose 4B column was used for the further purification. The column was activated with the CNBr and ovomucoid was coupled to the activated gel. This activated slurry was washed with the 0.1M NaHCO3 and 1M NaCl and then with water and finally with 1M NaCl and stored at 1M NaCl and 0.025% NaN3. The column was prepared and the sample was eluted with the same buffer. The fractions of the sample were collected at regular intervals and the presence of the protein was determined by performing the agglutinating assay. (Birck et al., 2004).The UV absorption method was followed for protein estimation. Almost 97% of the lectin was eluted and the remaining percentage present in the column was finally eluted using the 1M NaCl, 0.1M Sodium acetate buffer and water. These elute was dialysed and then concentrated to extract the protein. This method of extraction can give a yield of around 48 mg per 100g of the sample. The yield will vary based on the methodology adopted and the performance of the column. Affinity chromatography can be further used for the complete purification of the sample. The use of specific binding ligands for lectin can completely purify the lectin. Immobilized fetuin affinity chromatography was used by Kilpatrick and Yeoman for the purification of lectin in the year 1978. N-acetylglucosamine containing polysaccharides derived from the fungus Aspergillus niger was used for the purification of lectin by affinity chromatography by Kocourek and Horeisi by 1978.the D-galactose derived –sepharose was used for purification of lectin by Joubert et al. (1986). Recently two affinity columns having two different characteristics were used for the purification of the glycoprotein. One column containing the immobilized concavallin A (Con A) and the other containing Con-A Sepharose and ConA-XP3507 can be used for the purification. The nature of the nonspecific interactions between these two columns differs and leads to better purification of lectin. The ionic interactions are greater in the Con A sepharose column and hydrophobic interactions were greater at the Con A –XP3507 column. (Nilsen et al., 1990). These columns are more effective in purifying the lectin. The elution medium contained 1M NaCl and 20% ethylene glycol. This medium is very useful in desorbing nonspecifically bound materials. This method was very efficient at the room temperature. Thus Con A – Sepharose column was found to have greater purification efficiency than the other affinity chromatography. (Nilsen et al, 1990). (Scopes, 1994). Applications of lectin: 1. It is used for agglutination studies. 2. It is used for blood grouping studies. 3. It is used for lymphocyte sub- population studies. 4. It is used for histo chemical studies at normal and pathological studies. 5. It is used for the purification of the glycoprotein. 6. IT is used for the isolation of the recombinant proteins in transgenic plants 7. it is used for identifying the recombinant glycoproteins in the plants. 8. Some Lectin inhibit the bacterial growth in the rat gut by inhibiting the E.Coli that are sensitive to the mannose sugar. 9. Some of the Lectin are efficient against insect pests, nematode and mammalian pests. (Van Damme, 1998). References: Baues,RJ., and Gray, GR., 1976, “Lectin purification on Affinity columns containing reductively aminated disachharides”, Journal of Biological chemistry, Vol.252, No.1, 57-60. Birck, C., Damian, L., Marty-Detraves, C., Lougarre, A., Schulze-Briese, C., Koehl, C., Fournier, D., Paquereau, L., and Samama, JP., 2004, “ A New Lectin Family with Structure Similarity to Actinoporins Revealed by the Crystal Structure of Xerocomus chrysenteron Lectin XCL “, Journal of molecular biology, Vol. 344 , 1409–1420. Bonner, 2007, “Protein purification”, Routledge. Brooks, SA.,Leathem, AJC., and Schumacher,U., 1997, “Lectin Histochemistry: a concise practical Handbook”, BIOS Scientific in association with the Royal Microscopical Society. Desai, NN., Allen, A., and Neuberger, A., 1981, “Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages”, Biochemistry.Journal, Vol.197, 345-353. Dubey, RC.,2004, “A Textbook of Biotechnology”, 4th Edition, S. Chand and Company. Joubert, FJ., Sharon,N., and Merrifield,EH.,1986, “Purification and properties of a lectin from lonchocarpus capassa (apple-leaf) seed”, Phytochemistry, Vol. 25, No.2, 323 – 327. Sharon, N., and Lis, H., 2007, “Lectin”, Springer. Scopes, RK., 1994, “Protein purification: principles and practice”, Springer. Van Damme, EJM., 1998, “ Handbook of plant Lectin”, John Wiley and Sons. Read More